A Novel Poisson Distribution-Based Approach for Testing Boundaries of Real-Time PCR Assays for Food Pathogen Quantification

Abstract

The validation of quantitative real-time PCR systems and above all, proof of the detection limit of this method, is a frequently and intensively discussed topic in food pathogen detection. Among proper sample collection, assay design, careful experimental design, execution of real-time PCR, and data analysis, the validation of the method per se ensuring reliable quantification data is of prime importance. The purpose of this study was to evaluate a novel validation tool for real-time PCR assays, based on the theoretical possibility of the amplification of a single DNA target. The underlying mathematical basis for the work is Poisson distribution, which describes patterns of low particle numbers in a volume. In this context, we focused on the quantitative aspect of real-time PCR for the first time. This allowed for demonstration of the reliable amplification of a lone target DNA molecule and the demonstration of the distinct discrimination between integer molecular numbers when using low initial copy numbers. A real-time PCR assay amplifying a 274-bp fragment of the positive regulatory protein A locus of Listeria monocytogenes was used for this work. Evidence for a linear range of quantification from a single target copy to 10 ng of target DNA was experimentally demonstrated, and evidence for the significance of this novel validation approach is presented here.

Assessment of three commercial DNA extraction kits and a laboratory-developed method for detecting Cryptosporidium and Cyclospora in raspberry wash, basil wash and pesto

“…The Cp and standard deviations for the 1 copy dilutions were 41.0 ± 0.8 for the 18S rRNA assay and 37.1 ± 0.9 for the hsp70 assay. For all qPCR assays the percentage of positive reactions for the 1 copy dilution followed the Poisson distribution expected for low copy number solutions (Bustin et al, 2009;Rossmanith and Wagner, 2011) demonstrating reliable amplification of a single copy of target DNA under ideal conditions. It was necessary to identify a Cp value cut-off or boundary where samples with a Cp value above would be considered unreliable or negative.…”

A Novel Method for Sampling and Long-Term Monitoring of Microbes That Uses Stickers of Plain Paper

“…Swabbing was omitted in this setup, but three time periods were included. Since it was demonstrated that the prfA assay can detect and quantify even down to a single molecule (17), each positive signal in qPCR was rated as a positive result. The results summarized in Tables 2 and 3 show that stickers detected both bacterial species repeatedly from several locations, suggesting suitability as an appropriate on-site sampling/detection system.…”

“…A promising departure from growth-based methods is quantitative PCR (qPCR), which provides faster results. Theoretically, the detection limit of this method approaches one copy of the target gene (17). Practically, it is an especially sensitive tool that has undergone enormous developmental progress over the past several years (18).…”

How to evaluate PCR assays for the detection of low‐level DNA

“…The technique is capable of detecting a single DNA copy . And as the number of positive reactions follows the Poisson distribution , and each copy is counted in the Poisson distribution, this implies directly that each copy will be amplified in the real detection system.…”